SUMMARY: Influenza A viruses (IAVs) cause serious respiratory illness in humans, with ~250,000-500,000 deaths per year globally. In addition to seasonal epidemics, the ongoing pandemic threat posed by new, emerging, reassortant influenza viruses, for which humans are immunologically naive, represents a major public health concern. Current influenza vaccines are impacted by several issues. These include, the elicitation of narrow, strain-specific immune responses, an over-reliance on egg-based manufacturing methods, a protracted production process (>6 months), the need to predict in advance which strains will circulate in forthcoming seasons and the minimal induction of cellular and humoral immune responses to multiple influenza antigens (Ags) simultaneously. The sub-optimal performance of seasonal influenza vaccines in recent years has accelerated interest in developing a universal influenza virus vaccine, capable of providing broad and long- lived protection against seasonal and pandemic subtypes. Strategies to achieve this include refocusing immune responses towards highly conserved epitopes on influenza virus antigens such as the stalk of the major surface glycoprotein, hemagglutinin (HA), the neuraminidase (NA) or the internal nucleoprotein (NP). My research aims to develop an alternative, optimized, universal influenza vaccine platform which will overcome issues associated with current vaccines using three approaches. (1) Firstly, I will optimize polycistronic Ag expression cassettes, in which multiple IAV Ags are expressed simultaneously. These will include bi- or tri-cistronic Ag cassettes featuring headless HAs from group 1 or group 2 IAVs in combination with NA and/or NP. I will augment/broaden immune recognition of headless HA or NA by targeting Ags to host- derived extracellular vesicles (EVs) including exosomes in vivo. This will be achieved by engineering fusion-Ag constructs to tether Ag to a protein domain enriched in exosomes. Exosomes are nano-sized EVs which play important roles in the regulation of immune responses, due to their ability to present Ag, in addition to MHC and co-stimulatory molecules, to T- and B-cells. (2) Secondly, I will engineer these Ag constructs into non- replicating, rare species adenoviral (Ad) vectored vaccines, which have established protocols for clinical manufacturing, can be thermostabilized with minimal losses to immunogenicity under cold-chain free conditions and have demonstrated safety and immunogenicity in infants, adults and the elderly in clinical trials. (3) Finally, I will comprehensively evaluate and phenotype the magnitude and profile of these universal influenza vaccines in single-shot regimens. These data will provide valuable information for the design of subsequent prime:boost regimens and for challenge experiments in the future. In summary, the universal influenza vaccine platform described in this proposal would be well-suited to stockpiling for pandemic preparedness, and could provide heterologous protection following a single shot, which may be sufficient to ease the burden on the healthcare system in the early phase of an emerging pandemic.